Factor XIII is a newly identified binding partner for platelet collagen receptor GPVI‐dimer—An interaction that may modulate fibrin crosslinking

Abstract Background In the fibrin‐forming process, thrombin cleaves fibrinogen to fibrin, which form fibrils and then fibers, producing a gel‐like clot. Thrombin also activates coagulation factor XIII (FXIII), which crosslinks fibrin γ‐chains and α‐chains, stabilizing the clot. Many proteins bind to fibrin, including FXIII, an established regulation of clot structure, and platelet glycoprotein VI (GPVI), whose contribution to clot function is largely unknown. FXIII is present in plasma, but the abundant FXIII in platelet cytosol becomes exposed to the surface of strongly activated platelets. Objectives We determined if GPVI interacts with FXIII and how this might modulate clot formation. Methods We measured interactions between recombinant proteins of the GPVI extracellular domain: GPVI‐dimer (GPVI‐Fc2) or monomer (GPVIex) and FXIII proteins (nonactivated and thrombin‐activated FXIII, FXIII subunits A and B) by ELISA. Binding to fibrin clots and fibrin γ‐chain crosslinking were analyzed by immunoblotting. Results GPVI‐dimer, but not GPVI‐monomer, bound to FXIII. GPVI‐dimer selectively bound to the FXIII A‐subunit, but not to the B‐subunit, an interaction that was decreased or abrogated by the GPVI‐dimer–specific antibody mFab‐F. The GPVI‐dimer–FXIII interaction decreased the extent of γ‐chain crosslinking, indicating a role in the regulation of clot formation. Conclusions This is the first report of the specific interaction between GPVI‐dimer and the A‐subunit of FXIII, as determined in an in vitro system with defined components. GPVI‐dimer–FXIII binding was inhibitory toward FXIII‐catalyzed crosslinking of fibrin γ‐chains in fibrin clots. This raises the possibility that GPVI‐dimer may negatively modulate fibrin crosslinking induced by FXIII, lessening clot stability.

• GPVI-dimer binds to FXIII via the A-subunit and binds noncompetitively with FXIII to fibrin clots.

| INTRODUC TI ON
Platelet collagen-receptor glycoprotein VI (GPVI) occurs as both monomers and constitutively present homodimers, the functional form. 1,2 GPVI-dimers bind to exposed subendothelial collagen fibers in injured vessels, initiating a signaling cascade leading to platelet activation, aggregate formation, and thrombus formation. GPVI-dimer level is increased in activated platelets, 2 and GPVI-dimer clustering in activated platelets brings associated signaling molecules in closer proximity, enhancing signaling. 3 Activated platelets present a membrane surface complex that stimulates the coagulation reaction, increasing active thrombin production. 4 These multiple processes culminate in formation of a platelet thrombus, 5,6 which is stabilized by fibrin production due to thrombin generation on the surface of activated platelets.
GPVI has also been suggested to be a fibrin(ogen) receptor, and fibrin fiber formation from fibrinogen enhances collagen-induced platelet activation. 7,8 Whether GPVI can actually bind fibrinogen remains controversial. 9, 10 We recently demonstrated that GPVIdimers, not -monomers, bind to fibrin fibers in clots. 9 Both collageninitiated platelet activation and coagulation pathways are necessary to form a stable blood clot, comprising many platelets, fibrin fibers, and coagulation factors, ensuring effective hemostasis and wound repair. Clot retraction which contributes to wound closure depends on the integrin α IIb β 3 -fibrin interaction, which allows the platelet cytoskeleton to draw fibrin fibers together. 11 Coagulation factor XIII (FXIII) 12 was also reported to be involved in clot retraction, 13,14 although the mechanisms involved are yet not well defined.
Glycoprotein VI also binds to other proteins, including laminin, 15 adiponectin, 16 and extracellular matrix metalloproteinase inducer, 17 so during the course of our studies on the GPVI-fibrin interaction, we explored whether it interacts with FXIII, part of the plasma milieu in which thrombus formation occurs.
Plasma FXIII is an inactive tetramer of two A-and two B-subunits (FXIIIA 2 B 2 ), until it is cleaved by thrombin in the presence of calcium, which removes the activation peptide from subunit A, freeing it from the complex, and converting it to the active form (FXIIIAa, where "a" designates an active form) that functions as a transglutaminase. 18 Platelet cytosol also contains abundant FXIII as active FXIIIA 2 , which is further activated by thrombin to FXIIIAa. Platelet FXIII was reported to be involved in clot retraction, platelet spreading and adhesion (as reviewed in Muszbek et al. 18 ); and platelet FXIII was shown to crosslink several proteins to the platelet cytoskeleton, suggesting that it may be involved in platelet morphological change. 19 However, FXIII deficiency does not affect platelet aggregation, indicating that it does not contribute to this process. 14 Although FXIII is not exposed on the surface of resting platelets, it is surface expressed upon strong platelet activation. 12 FXIIIAa crosslinks fibrin γ-chains, forming γ-dimers (γγ), and further crosslinks γ-and α-chains, forming higher-molecular-weight products. Crosslinked fibrin clots would be more stable and more resistant to fibrinolysis.
Platelets have been shown to participate in numerous coagulation processes, including fibrin formation, and therefore in this study we asked whether fibrin-binding GPVI-dimer may interact with FXIII, found in both platelets and plasma. We demonstrate that GPVI-dimers specifically and directly interact with FXIII, and its inhibitory effect on γ-γ fibrin crosslinking suggests that this interaction may modulate fibrin clot integrity. Table 1 shows the FXIII proteins (and their abbreviations) used in this study and their affinity constants (K D ) for binding to GPVI-dimer. PK1 fibrinogen (FXIII-, plasminogen-, fibronectin-free; Enzyme Research Laboratories, UK); recombinant proteins of human GPVIextracellular domain: GPVI-Fc 2 (dimer) and GPVI ex (monomer), developed by Moroi and Jung, were previously described. 9 The following antibodies were used: rabbit monoclonal antibody against human IRDye 800CW anti-human antibody, IRDye 800CW anti-rabbit antibody, and IRDye 680RD anti-rabbit antibody (Li-Cor, Lincoln, NE, USA). were used for GPVI-Fc 2 (GPVI-dimer) and GPVI ex (GPVI-monomer) binding assays as previously described. 19 Bound GPVI-Fc 2 and GPVI ex were detected by 1G5/IRDye 800CW anti-mouse antibody and quantified using an Odyssey CLx fluorescence imaging system (Li-Cor).

| Immunoblotting analysis by clot assay
GPVI-Fc 2 and FXIII binding to fibrin fibers and its effects on fibrin crosslinking were analyzed using a fibrin clot assay. 9 Figure 3A

| GPVI-Fc 2 specifically binds to FXIII
GPVI-Fc 2 binds with higher affinity to crosslinked fibrin than to noncrosslinked fibrin, 9 suggesting that FXIII may affect its binding or crosslinked fibrin would show a higher affinity to GPVI-Fc 2 . An ELISA assay was used to determine whether GPVI-Fc 2 and FXIII proteins are capable of direct interaction, as shown in Figure 1A (relative levels of binding at fixed GPVI-Fc 2 concentration) and   Table 1). rh-FXIIIAa  In contrast, GPVI-monomer (GPVI ex ) does not bind to any form of FXIII ( Figure 1C) and binds only weakly to collagen-III, indicating that GPVI must be in its dimeric form to bind to FXIII. Figure 1D shows that although the binding of GPVI-Fc 2 to collagen is moderately inhibited by high concentration of the GPVI-dimerspecific antibody mFab-F (200 µg/mL), this concentration severely decreases or abrogates GPVI-Fc 2 binding to all the FXIII-related proteins. This suggests that FXIII may bind near the collagen-binding site of GPVI-Fc 2 since GPVI-Fc 2 , not GPVI ex , binds specifically to collagen fibers 2 and its binding to FXIII is also dimer-specific ( Figure 1C). Addition of ZED A108, FXIII-transglutaminase inhibitor (Zedira, Darmstadt, Germany), had no effect on GPVI-dimer binding to FXIII (data not shown), showing that the transglutaminase activity of FXIII is not involved in the binding interaction, suggesting that GPVI-dimer-bound FXIII may retain enzymatic activity for a small substrate like biotinamidopentylamine.

| FXIII binding to GPVI-Fc 2 abrogated by inhibitory GPVI-dimer-specific antibody
Our data indicate that the active form of GPVI, GPVI-dimer, exclusively binds to FXIII A-subunit and complex FXIIIA 2 B 2 , while showing little interaction with subunit B. Notably, FXIII subunit B, not subunit A, was reported to bind to fibrinogen and stimulate activation of FXIII on fibrin. 21 This means that platelet FXIIIA 2 could bind to GPVI-dimers since it is exposed on the cell surface of Recombinant (rh) Too low to be determined Note: The FXIII proteins were obtained from the following suppliers: a Fisher Scientific, Leicestershire, UK; and b Zedira, Darmstadt, Germany. Binding affinities (K D ) were calculated from the binding curves shown in Figure 1B; data are expressed as values of the mean ± SEM (n = 8). The binding affinity of the fully activated form of the FXIII A-subunit is comparable to GPVI-dimer's affinity to collagen type III (0.183 ± 0.026).
Abbreviations: FVIII, factor VIII; GPVI, glycoprotein VI; SEM, standard error of the mean. strongly activated platelets. 22,23 Mitchell et al 23 showed that platelet FXIII-induced fibrin crosslinking occurs when no plasma FXIII is present, suggesting that active FXIII is exposed on the platelet surface, performing its function to crosslink fibrin chains. FXIII of activated platelets localizes in sphingomyelin-rich rafts, 13 and GPVI was also present in rafts on the platelet membrane. 24 This raises the tantalizing possibility that GPVI-dimer on the platelet membrane may serve as a receptor for platelet FXIII. Since the A-subunit is an active subunit, the interaction with GPVI might also influence the transglutaminase activity of FXIII from plasma or platelets, modulating the crosslinking of fibrin clots.

| GPVI is not a principal substrate of FXIII
It can be hypothesized that FXIII might crosslink GPVI to fibrin since GPVI binds more to crosslinked fibrin than to noncrosslinked fibrin. 9 We tested this using reaction mixtures comprising GPVI-Fc 2 , FXIIIA 2 B 2 , thrombin (1 U/mL), 2 mM Ca 2+ , and biotinamidopentylamine in the presence or absence of FXIII-free fibrinogen and determined if the transglutaminase activity of FXIII can transfer the biotinyl moiety from biotinamidopentylamine to GPVI-dimer, which would indicate that GPVI-dimer is a substrate of FXIII. Figure 2A shows a western blot simultaneously stained for GPVI-Fc (green) and proteins with incorporated biotin (red). In the sample with a high FXIII concentration (100 µg/mL; lane 4, Figure 2A), an obvious biotinylated protein band is seen at ≈80 kDa, and there is only a very faint band on the position of GPVI-Fc that can be seen in  Table 1. (C) GPVI-Fc 2 (GPVI-dimer) and GPVI ex (GPVI-monomer) binding to FXIII proteins, with collagen-III as a positive control. GPVI-Fc 2 or GPVI ex was 50 µg/mL and the wells were coated with a FXIII protein or collagen type III (Col-III) at 10 μg/mL. The P value (n = 3) comparing GPVI-Fc 2 binding to GPVI ex binding for each FXIII protein is shown above each set of bars. GPVI-Fc 2 bound to col-III and all FXIII proteins except for FXIII B-subunit. GPVI ex did not bind to any of the FXIII proteins and only weakly bound to col-III. (D) Effects of GPVIdimer−specific antibody (mFab-F) on GPVI-Fc 2 binding to FXIIIA 2 B 2 , rh-FXIIIA 2 , rh-FXIIIAa (thrombin activated rh-FXIIIA 2 ), rh-FXIIIB 2 , and col-III. The P value (n = 4) comparing no mFab-F addition and addition of mFab-F (+fab) of each FXIII protein is shown above each set of bars.
The addition of mFab-F (200 µg/mL) dramatically decreased or abrogated the binding of GPVI-Fc 2 to all the FXIIIs. FVIII, factor VIII; GPVI, glycoprotein VI some GPVI-crosslinking may occur at high FXIII concentrations. In the fibrin clot, biotin was incorporated to fibrin α-chain, γ-γ dimer, and higher-molecular-weight bands, but little GPVI-Fc (green) was associated with bands with molecular weight higher than GPVI-Fc, suggesting that GPVI-Fc would not be crosslinked to fibrin under our experimental conditions where FXIII is used at concentrations of 1-5 μg/mL. This figure indicates that crosslinking occurred predominantly in the fibrin γ-and α-chains.

| GPVI-dimer-FXIII interaction in fibrin clots
We determined if FXIII might affect GPVI-dimer binding to fibrin fibers and vice versa using our clot binding assay, which determines  Figure 3D, adding FXIII at 1 μg/mL increases γ-dimer, and addition of GPVI-Fc 2 decreases it (middle set of bars), but this effect was not evident at higher FXIII concentration (rightmost set of bars). Since the enzymatic activity of FXIII is not involved in the binding to GPVI-Fc 2 , the inhibitory activity F I G U R E 3 Effects of GPVI-Fc 2 on the binding of FXIII and on γ-chain crosslinking. Each data value in all the graphs is the mean ± SEM (n = 4). (A) Schematic diagram of the fibrin clot assay, which we previously described in detail. 9 In graphs (B) and (C), clots were formed from FXIII-free fibrinogen, GPVI-Fc 2 (0, 25, 50, or 100 μg/mL), and FXIIIA 2 B 2 at 0 (•), 1 (■), or 5 (▲) μg/mL and the binding of GPVI-Fc to fibrin was determined. The amount of GPVI bound to fibrin (expressed as a percent of the control GPVI band (unclotted sample)) was not affected by FXIII. (C). The amount of FXIII bound to fibrin was not affected by GPVI-Fc 2 . The fluorescence of the FXIII band was reported as arbitrary units (AU). (D) Amount of fibrin γ-dimer (γγ, expressed as % of the original γ-band) in the clots formed in the presence of FXIIIA 2 B 2 (0, 1, or 5 μg/mL) in the absence or presence of 100 μg/mL GPVI-Fc 2 . In each two-bar set, the checkered bars show the effect of adding GPVI-Fc 2 to FXIII at the indicated concentration. The clots formed in the presence of FXIIIA 2 B 2 at 1 μg/mL show a significant decrease in γ-dimer (P = .0476). (E) Amount of γ-monomer (expressed as a percentage of the original γ-band) in the clots formed in the presence of FXIIIA 2 B 2 (0, 1, or 5 μg/mL) in the absence or presence of 100 μg/mL GPVI-Fc 2 . In each two-bar set, the checkered bars show the effect of adding GPVI-Fc 2 to FXIII at the indicated concentration. Adding GPVI-Fc 2 had no significant effect on the amount of γ-monomer at any FXIIIA 2 B 2 concentration. FVIII, factor VIII; GPVI, glycoprotein VI; SEM, standard error of the mean of GPVI-Fc 2 would be due to steric hinderance. In contrast, adding GPVI-Fc 2 had no significant effect on the amount of γ-monomer ( Figure 3E).

| CON CLUS ION
Using an in vitro system of defined components, we demonstrated for the first time that GPVI-dimer specifically interacts with the FXIIIAa (activated A-subunit of FXIII), and FXIII and GPVI-dimer independently bind to fibrin at proximal sites in the fibrin D-domain.
This suggests that the binding site for FXIII on GPVI-dimer is different from its fibrin-binding site and the binding site for GPVI-dimer on FXIIIA 2 is different from its fibrin-binding site. We can hypothesize that the specific binding of GPVI-dimer to FXIIIAa demonstrated in our study may negatively modulate the crosslinking activity of FXIIIAa and thus affect clot stability. The occurrence of this interaction between GPVI-dimer and FXIII in the physiological context, in plasma and/or FXIII expressed on the activated platelet, and its function must be assessed in future work.

R EL ATI O N S H I P D I SCLOS U R E
None of the authors have any conflicts of interests to report.